| Ginkgo Semen is the dried seeds of Ginkgo biloba L.,which was first recorded in Ri-Yong-Ben-Cao.Ginkgo Semen is a conventional Chinese medicine which is widely used to treat cough and asthma.It possesses the effects of astringing lung for relieving cough and asthma.It is often used to treat phlegm,cough,and asthma.To our knowledge,there are few studies about the active ingredients accounting for the antitussive effect of Ginkgo Semen Based on the antitussive effect of Ginkgo Semen,(2-(1-((27R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-indol-3-yl)acetyl)-L-aspartic acid(GK-A)had been isolated from Ginkgo Semen in our laboratory,previously.GK-A showed a significant antitussive effect based on pharmacodynamic evaluation results,which indicated that GK-A showed the potential for new antitussive drugs development.In order to elucidate the disposal process of GK-A in vivo,the pharmacokinetics,intestinal absorption,tissue distribution,metabolism,and excretion characteristics of GK-A in vivo and in vitro were systematically studied in this research.This research will lay the experimental foundation for the in-depth research and the development of new antitussive drugs of GK-A and Ginkgo Semen.1.Isolation and purification of GK-AFirstly,the active section with high content of GK-A in Ginkgo Semen was purified by macroporous resin.Then the active section was further separated and purified by preparative chromatography,and approximate 5 g of GK-A with purity more than 98%was harvested,which provided a sufficient sample for the following pharmacokinetics of GK-A and other related studies.2.The pharmacokinetic study of GK-AUPLC-MS/MS was used to determine the content of GK-A in rat plasma.Isoquercetin was used as internal standard,and the electrospray(ESI)ion source was used to determine the content of GK-A in the positive ion mode with multiple reaction monitoring(MRM)mode.The results showed that the calibration curve showed a good relationship in the range of the concentration of 37.5-7500.0 ng·ml-1 with correlation coefficient(r2)higher than 0.99.The lower limit of quantification(LLOQ)was 37.5 ng·mL-1,and the extraction recovery was more than 85%.This method also possessed a good the specificity,and the precision,accuracy,and stability met the requirements of biological sample analysis without obvious matrix effect.Rats were given 15,30,60 mg·kg-1 GK-A by intragastrical administration and 1 mg·kg-1 GK-A by intravenous injection for the pharmacokinetic study,respectively.The plasma was obtained at different time points after the administration,and the concentration of GK-A in plasma was measured by established method.The pharmacokinetic parameters were calculated by DAS 3.0 and the absolute bioavailability was also calculated.The Tmax of GK-A at 15,30,60mg·kg-1 were 1.667±0.683,1.500±0.447,1.500±0.447h,and the half-life(T1/2)of GK-A at 15,30,60 mg·kg-1 were 0.429±0.089,0.682±0.571,1.619±1.271 h,respectively.The T1/2 of GK-A after the intravenous injection was 0.500±0.037 h.The results showed that GK-A was absorbed slowly and eliminated rapidly in rats,and its absolute bioavailability was 1.83±0.09%.3.The absorption of GK-AFirstly,the stability of GK-A in simulated gastrointestinal condition was studied by HPLC.The results showed that GK-A kept stable in different pH buffer solutions(pH 1.2,6.8,7.4),artificial gastric juice,artificial intestinal juice,and intestinal contents,but GK-A partially degraded when incubated with the intestinal contents of rats.Then,the absorption features of GK-A in different intestinal segments of rats was studied by single-pass perfusion model.The concentration of GK-A in perfusion solution at different time points was determined by HPLC,and the absorption rate constant(Ka)and the apparent absorption coefficient(Peff)were calculated.The results showed that GK-A could be absorbed in each intestinal segment of rats with no specific absorption site.There was no significant difference of the absorption of GK-A in the perfusion solution at pH 6.5 and 7.4.The absorption parameters of GK-A would not be significantly changed when verapamil,a P-glycoprotein inhibitor,and indomethacin,a multidrug-resistant protein 2 inhibitor,were added in the perfusion solution,indicating that GK-A was not the substrate of P-glycoprotein and multidrug-resistant protein4.The tissue distribution of GK-A.Firstly,the binding rate of plasma protein in rats of GK-A was studied.A method for the determination of GK-A in phosphate buffer(PBS)was established by UPLC-MS/MS Isoquercetin was used as internal standard,and the content of GK-A in ultrafiltrate of plasma was determined by a mass spectrometer equipped with electrospray ionization(ESI)ion source operating in positive ion mode with multiple reaction ion monitoring(MRM)mode The results showed that the calibration curve exhibited a satisfactory linearity over the range of 25.0-2500.0 ng·mL-1 with r2 higher than 0.99,and the lower limit of quantification was 2 5 ng·mL-1.The extraction recovery was greater than 8 5%,and the specificity of the method was satisfactory.The precision,accuracy,and stability met the requirements of biological sample analysis.The results showed that the binding rate of plasma protein of GK-A was 34.0±2.2%,which belonged to low-intensity binding,and the binding rate of plasma protein would not change within the concentration range of the study.Then,a UPLC-MS/MS method was established to determine the concentration of GK-in rat tissue homogenate(heart,liver,spleen,lung,kidney,brain,and stomach).(2-(1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-indol-3-yl)acetyl)-L-glutamic acid(GK-2)was selected as the internal standard.The content of GK-A in each tissue homogenate was determined by a mass spectrometry equipped with an electrospray ion(ESI)source operating in positive ion mode with multiple reaction monitoring(MRM)mode.The results showed that the calibration curves exhibited a satisfactory linearity in the range of 0.018-4.500 ?g·mL-1,with r2 greater than 0.99,and the lower limit of quantification was 0.018?g·mL-1.The recoveries of GK-A in all tissue homogenate were higher than 70%without obvious matrix effects.Precision,accuracy,and stability met the requirements of biological analysis.The content of GK-A in different tissue homogenate was determined after the intravenous administration of GK-A of 1 mg·kg-1 in rats.The results showed that GK-A could be detected in all tissues except brain tissue at 0.5 h after administration.The peak concentration of GK-A was reached at 0.5 h after the administration,and the highest concentration of GK-A was occurred in kidney.Because GK-A could not be detected in brain tissue,it was indicated that GK-A could not penetrate blood-brain barrier and be distributed in brain.GK-A was eliminated rapidly in tissues,and it was almost eliminated from all tissues except liver and kidney within 6 h after the administration,indicating that GK-A was not accumulated in tissues.Our result showed that GK-A might be mainly excreted from the kidney after absorption into the blood.5.The metabolism of GK-A.Firstly,we summarized the possible metabolites of GK-A according to the common metabolic types of drugs.Then,metabolic characteristics of GK-A was studied by UPLC-QTOF-MS/MS in vivo and in vitro.Four metabolites of GK-A were identified according to the fragmentation features of GK-A in mass spectrometry.The metabolic reaction types of GK-A were including deglycosylation,hydroxylation,methylation,and hydrolysis.6.The excretion of GK-AAn established UPLC-MS/MS method was used for the determination of GK-A in rat bile and urine.GK-2 was used as the internal standard.The content of GK-A in rat bile and urine was determined by a mass spectrometry equipped with an electrospray ion(ESI)source operating in positive ion mode with multiple reaction monitoring(MRM)mode.The results showed that the calibration curves of GK-A exhibited a satisfactory linearity over the range of concentration of 0.12-15.00 ?g·mL-1 with r2 higher than 0.99,and the lower limit of quantification was 0.12 ?g·mL-1.The extraction recoveries were more than 85%.The methods were specific for the determination of GK-A without obvious matrix effects.The precision,accuracy,and stability met the requirements of biological sample analysis.The cumulative excretion rate of GK-A in urine of rats was 0.58±0.26%within 72 h after the administration,and the cumulative excretion rate in bile of rats was 0.95±0.27%within 24 h after administration.Then,the main metabolite MS1 of GK-A was synthesized by chemical synthesis.Then the contents of GK-A and MS1 in rat feces were determined by UPLC-MS/MS.GK-2 was used as internal standard.The contents of GK-A and MS1 in rat feces and urine were determined by a mass spectrometry was equipped with an electro spray ion(ESI)source operating in positive ion mode with multiple reaction monitoring(MRM)mode.The results showed that the calibration curves of GK-A and MS1 displayed a good linear relationship in the concentration range of 0.12-30 ?g·mL-1 with 11 higher than 0.99,and the lower limit of quantification was 0.12 ?g·mL-1.The extraction recovery was more than 85%with a satisfactory specificity.The precision,accuracy,and stability met the requirements of biological sample analysis without obvious matrix effect.After the intragastrical administration of GK-A in rats at a dose of 15 mg·kg-1,the cumulative excretion rate was calculated according to the measured concentration,and the cumulative excretion rate of GK-A in rat feces was 59.83±6.48%within 0-72 h after the administration,which indicated that GK-A was mainly excreted in feces.In conclusion,GK-A,an antitussive compound isolated from Ginkgo Semen,possesses a low absolute bioavailability.GK-A could keep stable in the simulated gastrointestinal environment,and GK-A will undergo some degradation when incubated with contents from the large intestine.GK-A can be absorbed in all intestinal segments of rats with an absorption rate.GK-A possessed a low binding rate of plasma protein.GK-A is widely distributed in various tissues except brain tissue of rats,and most of GK-A is distributed in kidney.GK-A could not be detected in the brain tissue of rats,indicating that it is difficult for GK-A to pass blood-brain barrier.GK-A is mainly metabolized by intestinal flora.After the oral administration,GK-A is mainly excreted in feces. |
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